Stabilized phenol nitroprusside reagent and analysis of nitrogen

ABSTRACT

An aqueous solution of phenol and nitroprusside is stabilized with a chelating agent such as EDTA or NTA. The reagent is particularly useful for the determination of urea nitrogen by a modified urease-Berthelot reaction.

United States Patent [1 1 Bressler et a1.

[ Oct. 30, 1973 STABILIZED PIIENOL NITROPRUSSIDE REAGENT AND ANALYSIS OF NITROGEN [7 51 Inventors: Leo F. Bressler, St. Louis; Charles F. Steinbrink, Jr., Fenton, both of [21] Appl. No.: 248,873

[52] U.S. Cl. 195/1035 R, 195/99, 23/230 B [51] Int. Cl. G0ln 31/14 [58] Field of Search 195/1035 R;

[56] References Cited UNlTED STATES PATENTS 3,119,751 1/1964 Chaney 195/1035 R 3,432,395 3/1969 Reardon 195/1035 R FOREIGN PATENTS OR APPLICATIONS 872,933 6/1971 Canada 195/1035 R OTHER PUBLICATIONS Henry Clin. Chem: Principles and Technics" p. 266-269 (1964).

Primary Examiner-Alvin E. Tanenholtz Assistant ExaminerMax D. Hensley Attorney-Philip B. Polster et a1.

[5 7] ABSTRACT An aqueous solution of phenol and nitroprusside is stabilized with a chelating agent-such as EDTA or NTA. The reagent is particularly useful for the determination of urea nitrogen by a modified urease- Berthelot reaction.

7 Claims, No Drawings STABILIZED PHENOL NITROPRUSSIDE REAGENT AND ANALYSIS OF NITROGEN BACKGROUND OF THE INVENTION This invention relates to a stabilized phenol nitroprusside reagent useful, for example, in the determination of urea nitrogen by urease hydrolysis of urea to ammonia and colorimetric determination of ammonia by a Berthelot method utilizing the phenolnitroprusside reagent and an alkaline hypochlorite reagent. The method of Fawcett and Scott, J. Clin. Path., 13, 156 (1960) and a modification of the method described by Chaney in US. Pat. No. 3,119,751 have provided a useful approach to the determination of urea nitrogen in biological fluids and ammonia nitrogen in other solutions. However, these approaches have required that fresh reagents be made up relatively frequently, because the required aqueous solutions of phenol (or phenate) and nitroprusside are well known to be unstable.

The present invention further simplifies the determination of urea nitrogen by providing a stable aqueous solution of phenol and nitroprusside.

SUMMARY OF THE INVENTION water white appearance of the reagent upon long term storage. The reagent may be used in a procedure in which urea nitrogen is determined in a biological fluid by mixing the sample and a lyophilized urease buffer to hydrolize urea to ammonia, the stabilized phenol nitroprusside reagent and an alkaline hypochlorite reagent are added to develop a blue green color, and the depth of color is determined at a wave length of from 500 to 650 mg.

DESCRIPTION OF THE PREFERRED EMBODIMENT The following examples are illustrative of stable reagents of the present invention:

EXAMPLE 1 56 grams of phenol 0.25 grams of sodium nitroprusside 0.020 grams EDTA 1000 ml water i The foregoing reagents are thoroughly mixed an stored in an amber bottle at to C. The reagent retains its water white appearance for at least six months.

EXAMPLE 2 56 grams phenol 0.25 grams nitroprusside 0.020 grams NTA (nitrilotriacetic acid) 1000 ml water EXAMPLE 3 56 grams phenol 0.25 grams nitroprusside 0.020 grams EGTA (ethylene glycol-bis-B-amino ethyl ether N,N' tetraacetic acid) 1000 ml water EXAMPLE 4 56 grams phenol 0.25 grams nitroprusside I 0.020 grams ethylene diamine di(o-hydroxy-phenyl acetic acid) 1000 ml water In all of the foregoing Examples 2-4 the degradation of the reagent by the formation of a blue color is inhibited markedly as compared with a reagent without the chelating agent, stored under like conditions.

As an example of the use of the stabilized reagent of the invention, the following procedure is given:

Three cuvettes (ex. 19X 105 mm) are labeled blank, standard and test and to each cuvette is 20 added 0.5 ml urease solution (3 units per ml). To the blank tube is added 11.1 water, to the standard" tube is added 10 #1 urea nitrogen standard solution (30 mg per 100 ml water), and to the test" tube is added 10 p.l of plasma. The tubes are incubated for 5 to 10 minutes at 37 or for to minutes at room temperature to hydrolize urea to ammonia. To each tube is added one ml phenol nitroprusside solution (from EX- AMPLE 1), 1 ml alkaline hypochlorite reagent (2.1 gm sodium hypochlorite, gm NaOH in 1000 ml water), and 5 ml water. The reagents are mixed and incubated at 37 C'for at least 5 minutes or at room temperature for at least 20 minutes. Optical density is read at 570 mp. (plus or' minus 10 mp). Urea nitrogen concentration'(mg%) of the test solution is equal to times the ratio of optical density of the test solution to the optical density of the standard solution.

The same procedure may be used to determine urinary urea nitrogen levels by substituting 10 micro liters of a 100 fold dilution of urine for the serum of the fore-' 40 going procedure.

It will be seen that the nitrogen concentration may be determined from a prepared calibration curve; however, such a curve should be redone for each new lot of phenol nitroprusside reagent and alkaline hypochlorite reagent.

Having thus described the invention, what is claimed and desired to be secured by Letters Patent is:

1. A'reagent consisting of an aqueous solution of phenol, an alkaline metal salt of nitroprussic acid, and sufof the solution.

2. The reagent of claim 1 wherein the phenol concentration is approximately 5-7 percent (W/V), the nitroprusside is between about 0.2 and 4.0 millimolar and the chelating agent is between 10 and 10 molar. 3. The reagent of claim 1 wherein the chelating agent is EDTA.

4. The reagent of claim 1 wherein the chelating agent is NTA.

5. A reagent package for the determination of urea nitrogen comprising a first vial of buffered urease; a second vial containing a reagent consiting of an aqueous solution of phenol, an alkaline metal salt of nitroprussic acid and a sufficient amount of a chelating agent to inhibit colaration of the solution of phenol and alkaline metal salt of nitroprussic acid; and a third vial of an aqueous solution of alkaline metal hypochlorite.

ficient amount of a chelating agent to inhibit coloration 6. A method for the determination of ammonia nitrogen in a test solution comprising adding to the test solution an alkaline hypochlorite reagent and a stabilized phenol nitroprusside reagent, said stabilized phenol nitroprusside reagent consisting essentially of an aqueous solution of phenol, an alkaline metal salt of nitroprussic acid, and sufficient amount of a chelating agent to inhibit coloration of the phenol nitroprusside reagent, and thereafter determining the depth of color formed, hence the nitrogen concentration in the test solution.

7. A method for determining the concentration of urea nitrogen in a biological fluid comprising a first step of urease hydrolysis of the urea nitrogen to convert 

2. The reagent of claim 1 wherein the phenol concentration is approximately 5-7 percent (W/V), the nitroprusside is between about 0.2 and 4.0 millimolar and the chelating agent is between 10 5 and 10 1 molar.
 3. The reagent of claim 1 wherein the chelating agent is EDTA.
 4. The reagent of claim 1 wherein the chelating agent is NTA.
 5. A reagent package for the determination of urea nitrogen comprising a first vial of buffered urease; a second vial containing a reagent consisting of an aqueous solution of phenol, an alkaline metal salt of nitroprussic acid and a sufficient amount of a chelating agent to inhibit coloration of the solution of phenol and alkaline metal salt of nitroprussic acid; and a third vial of an aqueous solution of alkaline metal hypochlorite.
 6. A method for the determination of ammonia nitrogen in a test solution comprising adding to the test solution an alkaline hypochlorite reagent and a stabilized phenol nitroprusside reagent, said stabilized phenol nitroprusside reagent consisting essentially of an aqueous solution of phenol, an alkaline metal salt of nitroprussic acid, and sufficient amount of a chelating agent to inhibit coloration of the phenol nitroprusside reagent, and thereafter determining the depth of color formed, hence the nitrogen concentration in the test solution.
 7. A method for determining the concentration of urea nitrogen in a biological fluid comprising a first step of urease hydrolysis of the urea nitrogen to convert the urea nitrogen in said biological fluid to ammonia nitrogen in solution, thereafter a second step of adding to said solution an alkaline hypochlorite reagent and a stabilized phenol nitroprusside reagent, said phenol nitropRusside reagent consisting essentially of an aqueous solution of phenol, an alkaline metal salt of nitroprussic acid and a sufficient amount of a chelating agent to inhibit coloration of the solution of phenol and alkaline metal salt of nitroprussic acid, and thereafter a third step of determining the depth of color formed, hence the concentration of urea nitrogen in the biological fluid. 